Journal: Molecular oncology

Article Title: Unveiling mutational dynamics in non-small cell lung cancer patients by quantitative EGFR profiling in vesicular RNA.

doi: 10.1002/1878-0261.12976

Figure Lengend Snippet: Fig. 1. Experimental validation of the NBI-ddPCR method. (A) Overview of the NBI experimental protocol and study workflow for high- sensitivity detection of EGFR activating and resistance mutations on plasma clinical samples. EVs were isolated from patient samples collected at baseline, first clinical evaluation (C3D1), and progression, and compared with available ctDNA and tissue biopsy analysis. Plasma-isolated EVs were directly analyzed through one-step ddPCR with mutation-specific fluorescent probes that could detect in a single assay the presence of both the WT and mutated EGFR mRNAs. Prior to mutation detection by ddPCR, a fraction of the isolated EVs were subjected to immunoprecipitation with anti-EGFR antibody beads to potentially enrich for tumor-derived EVs. (B) Validation of the one-step ddPCR EGFR mutation detection assay on EVs isolated from NSCLC cells by NBI (n = 3, mean with SD). No statistical test was applied for comparison. (C) Validation of the one-step ddPCR EGFR mutation detection assay on EVs isolated from plasma samples of healthy individuals by NBI. Number of mutated EGFR copies on EV-RNA obtained by NBI-ddPCR is represented as the number of copies/105 EVs.

Article Snippet: The screen assay was carried out in 384-OptiPlate (Perkin Elmer, Waltham, MA, USA) in a final volume of 20 lL using 105–106 vesicles as template, 15 lg mL 1 of Nickel Chelate AlphaLISA Acceptor Beads (Perkin Elmer), 10 lg mL 1 of AlphaScreen Streptavidin Donor beads (Perkin Elmer) and 20 ng of biotinylated anti-EGFR antibody (OriGene, Rockville, MD, USA).

Techniques: Biomarker Discovery, Clinical Proteomics, Isolation, Mutagenesis, Immunoprecipitation, Derivative Assay, Detection Assay, Comparison

Journal: Molecular oncology

Article Title: Unveiling mutational dynamics in non-small cell lung cancer patients by quantitative EGFR profiling in vesicular RNA.

doi: 10.1002/1878-0261.12976

Figure Lengend Snippet: Fig. 2. Detection of EGFR mutations in tumor tissue and sensitivity by liquid biopsy. (A) Rate of co-occurrence of multiple mutations (activating and resistance) at baseline, as measured by NBI-ddPCR (numbers in parenthesis indicate the relative number of cases over total, n = 27; WT indicates samples negative for any type of mutation tested; percentage values have been rounded). (B) Comparison of liquid biopsy by EV-RNA NBI-ddPCR with canonical ctDNA (qPCR) analysis for the detection of E746_A750del, L858R, and T790M mutations, shown as percentage of concordance (referred as sensitivity in detecting positivity to EGFR mutations) with the corresponding tissue biopsy (numbers in parenthesis indicate the number of patients that had matched results between tissue and liquid biopsy, n = 27). No statistical test was applied for comparison. (C) Frequency of patients with EGFR-activating and T790M resistance mutations detected in tissue and liquid biopsy, at baseline, first clinical evaluation (C3D1), and progression (other mutations: L747_S752del, E746-S752>V, E746-T751del, G719S, L833V, L861Q, L858M, exon 20 insertion; WT indicates samples negative for any type of mutation tested). Only E746_A750del, L858R, and T790M were tested on EV-RNA following NBI-ddPCR. Sum of percentages may exceed 100% of total because of rounding and concomitant presence of multiple mutations in the same patients (n = 27; ctDNA values obtained by mean of qPCR). No statistical test was applied for comparison (D) Difference in detected fractions of positive cases with EGFR-activating and T790M resistance mutations from C3D1 to progression, as measured by EV-RNA liquid biopsy (numbers in parenthesis indicate the relative number of cases over total, n = 27; percentage values have been rounded).

Article Snippet: The screen assay was carried out in 384-OptiPlate (Perkin Elmer, Waltham, MA, USA) in a final volume of 20 lL using 105–106 vesicles as template, 15 lg mL 1 of Nickel Chelate AlphaLISA Acceptor Beads (Perkin Elmer), 10 lg mL 1 of AlphaScreen Streptavidin Donor beads (Perkin Elmer) and 20 ng of biotinylated anti-EGFR antibody (OriGene, Rockville, MD, USA).

Techniques: Mutagenesis, Comparison

Journal: Molecular oncology

Article Title: Unveiling mutational dynamics in non-small cell lung cancer patients by quantitative EGFR profiling in vesicular RNA.

doi: 10.1002/1878-0261.12976

Figure Lengend Snippet: Fig. 3. Tumor heterogeneity and high frequency of T790M mutation by testing EGFR in ctDNA and EV-RNA. Intra-patient heterogeneity of EGFR mutations and levels of T790M detected by liquid biopsy analysis at baseline and progression. At baseline, analysis of ctDNA was performed by both qPCR and ddPCR. Only samples tested for E746_A750del, L858R, and T790M are included (n = 27; WT indicates samples negative for any type of mutation tested). Number of mutated EGFR copies on EV-RNA obtained by NBI-ddPCR is represented as linear multiple of 1010 copiesmL1.

Article Snippet: The screen assay was carried out in 384-OptiPlate (Perkin Elmer, Waltham, MA, USA) in a final volume of 20 lL using 105–106 vesicles as template, 15 lg mL 1 of Nickel Chelate AlphaLISA Acceptor Beads (Perkin Elmer), 10 lg mL 1 of AlphaScreen Streptavidin Donor beads (Perkin Elmer) and 20 ng of biotinylated anti-EGFR antibody (OriGene, Rockville, MD, USA).

Techniques: Mutagenesis

Journal: Molecular oncology

Article Title: Unveiling mutational dynamics in non-small cell lung cancer patients by quantitative EGFR profiling in vesicular RNA.

doi: 10.1002/1878-0261.12976

Figure Lengend Snippet: Fig. 4. Longitudinal analysis of EGFR mutations in NSCLC patients revealed by vesicular RNA. Longitudinal measurements of EV profiles were taken at baseline, first clinical evaluation (C3D1), and progression, alongside to tumor dimensional changes. (A) Box and whiskers distribution (median, min to max) of tumor size determined by the sum of longest diameters (SLD) and compared between baseline and C3D1 (n = 23; *P > 0.05, Wilcoxon signed-rank test, two-tailed) and between progression and C3D1 (n = 22; n.s., not significant, Wilcoxon signed-rank test, two-tailed). (B) Total EV count, represented as Log10 conversion of 1010 copies per mL of plasma, and EV size quantified by TRPS. (C) Number of mutated EGFR copies on EV-RNA as measured by NBI-ddPCR and represented as Log10 conversion of 1010 copiesmL1 (WT indicates samples negative for any type of mutation tested). (D) Number of mutated EGFR copies on EV-RNA as measured by NBI-ddPCR, represented as Log10 conversion of 105 copiesmL1, and quantified upon immunoprecipitation (IP) against the membrane EGFR protein on isolated EVs. Scatter plots of individual value in (B–D) are represented as mean with SD (*P > 0.05, **P < 0.005, ***P < 0.001, ****P < 0.0001; n.s: not significant; Wilcoxon signed-rank test, two-tailed).

Article Snippet: The screen assay was carried out in 384-OptiPlate (Perkin Elmer, Waltham, MA, USA) in a final volume of 20 lL using 105–106 vesicles as template, 15 lg mL 1 of Nickel Chelate AlphaLISA Acceptor Beads (Perkin Elmer), 10 lg mL 1 of AlphaScreen Streptavidin Donor beads (Perkin Elmer) and 20 ng of biotinylated anti-EGFR antibody (OriGene, Rockville, MD, USA).

Techniques: Two Tailed Test, Clinical Proteomics, Mutagenesis, Immunoprecipitation, Membrane, Isolation

Journal: Molecular oncology

Article Title: Unveiling mutational dynamics in non-small cell lung cancer patients by quantitative EGFR profiling in vesicular RNA.

doi: 10.1002/1878-0261.12976

Figure Lengend Snippet: Fig. 6. Association of NBI-ddPCR analysis with clinical features and patient outcome. (A) Distribution of the changes in the ratio of total number of EVs between the first clinical evaluation (C3D1) to progression in relation to patient survival status. Differences between the two groups are not significant (unpaired two-tailed t-test; data are represented as mean with SD). (B) Box and whiskers distribution (median, min to max) of radiologic PD, defined by RECIST 1.1, in association with the survival status (Alive, n = 8; Dead, n = 14; ****P < 0.0001, unpaired two-tailed t-test). (C) Box and whiskers representation (median, min to max) of radiologic PD, defined by RECIST 1.1, as divided into quartile distributions of groups of patients (Q1, n = 7; Q2, n = 5; Q3, n = 5; Q4, n = 6; n.s., not significant, *P < 0.05, ****P < 0.0001; one-way ANOVA with Tukey’s comparison test; data are represented as mean with SD). (D) Distribution of the changes in the ratio of total number of EVs between the first clinical evaluation (C3D1) to progression in the four radiologic PD quartile groups as defined in (C). Differences in the four groups are not significant (one-way ANOVA with Tukey’s comparison test). (E) Representative CT scan images of selected patients at the three clinical time points with parallel detection of the driver E746_A750del (orange circles) and the resistance T790M (purple circles) EGFR mutations in liquid biopsy (EV-RNA and ctDNA), as compared to tissue analysis. The largest target lesion is pointed out (yellow arrows) in the upper panel, while the volume of all lesions is represented as the SLD. Analysis of cfDNA was performed by qPCR; number of mutated EGFR copies on EV-RNA obtained by NBI-ddPCR is represented as multiple of 1010 copiesmL1. WT: wild- type samples for the indicated mutation.

Article Snippet: The screen assay was carried out in 384-OptiPlate (Perkin Elmer, Waltham, MA, USA) in a final volume of 20 lL using 105–106 vesicles as template, 15 lg mL 1 of Nickel Chelate AlphaLISA Acceptor Beads (Perkin Elmer), 10 lg mL 1 of AlphaScreen Streptavidin Donor beads (Perkin Elmer) and 20 ng of biotinylated anti-EGFR antibody (OriGene, Rockville, MD, USA).

Techniques: Two Tailed Test, Comparison, Computed Tomography, Mutagenesis